RESUMO
AIM: To compare the composition of the salivary microbiota in caries-affected vs. caries-free mutans streptococci (MS)- positive children with mixed dentition. MATERIALS AND METHODS: Twenty eight healthy, 11-12-year-old schoolchildren with high MS counts (>10â5 CFU/mL) were included in this study. The children were screened with the Dentocult SM Strip Mutans test (Orion Diagnostica, Espoo, Finland) and examined using the International Caries Detection and Assessment System (ICDAS). The microbial composition of the saliva was assessed using the Human Oral Microbe Identification Microarray (HOMIM). Microbial differences between caries-affected (n=18) and caries-free children (n=10) were compared by Mann-Whitney analysis. RESULTS: The microbiota of the caries-affected vs. caries-free children was rather similar. Abiotrophia defectiva and Actinomyces meyeri/A. odontolyticus were significantly higher in caries-affected than in caries-free children (p=0.006, 0.046, respectively). Shuttleworthia satelles was significantly higher in caries-free compared to caries-affected children (p=0.031). A. defectiva and A. meyeri/A. odontolyticus correlated positively with caries severity measured by ICDAS Caries Index (p = 0.494, 0.454, 0.400 respectively) while S. satelles was negatively correlated with caries severity (p= -0.489). CONCLUSIONS: Salivary A. defectiva and A. meyeri/A. odontolyticus and are associated with caries occurrence in MS-positive children with mixed dentition.
Assuntos
Cárie Dentária/microbiologia , Saliva/microbiologia , Streptococcus mutans/isolamento & purificação , Abiotrophia/isolamento & purificação , Actinomyces/classificação , Actinomyces/isolamento & purificação , Actinomycetaceae/classificação , Actinomycetaceae/isolamento & purificação , Carga Bacteriana , Carnobacteriaceae/isolamento & purificação , Criança , Índice CPO , Dentição Mista , Gemella/isolamento & purificação , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Streptococcus/classificação , Streptococcus/isolamento & purificaçãoRESUMO
Enterococcus faecalis strains secrete multiple peptides representing different sex pheromones that induce mating responses by bacteria carrying specific conjugative plasmids. The pheromone cAM373, which induces a response by the enterococcal plasmid pAM373, has been of interest because a similar activity is also secreted by Streptococcus gordonii and Staphylococcus aureus. The potential to facilitate intergeneric DNA transfer from E. faecalis is of concern because of extensive multiple antibiotic resistance, including vancomycin resistance, that has emerged among enterococci in recent years. Here, we characterize the related pheromone determinant in S. gordonii and show that the peptide it encodes, gordonii-cAM373, does indeed induce transfer of plasmid DNA from E. faecalis into S. gordonii. The streptococcal determinant camG encodes a lipoprotein with a leader sequence, the last 7 residues of which represent the gordonii-cAM373 heptapeptide SVFILAA. Synthetic forms of the peptide had activity similar to that of the enterococcal cAM373 AIFILAS. The lipoprotein moiety bore no resemblance to the lipoprotein encoded by E. faecalis. We also identified determinants in S. gordonii encoding a signal peptidase and an Eep-like zinc metalloprotease (lspA and eep, respectively) similar to those involved in processing certain pheromone precursors in E. faecalis. Mutations generated in camG, lspA, and eep each resulted in the ablation of gordonii-cAM373 activity in culture supernatants. This is the first genetic analysis of a potential sex pheromone system in a commensal oral streptococcal species, which may have implications for intergeneric gene acquisition in oral biofilms.
Assuntos
DNA Bacteriano/genética , DNA Intergênico/genética , Oligopeptídeos/metabolismo , Streptococcus gordonii/genética , Streptococcus gordonii/metabolismo , Sequência de Aminoácidos , Enterococcus faecalis/genética , Transferência Genética Horizontal/genética , Transferência Genética Horizontal/fisiologia , Dados de Sequência Molecular , Mutação , Oligopeptídeos/química , Oligopeptídeos/genética , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
Conjugative transposons are highly ubiquitous elements found throughout the bacterial world. Members of the Tn916-Tn1545 family carry the widely disseminated tetracycline-resistance determinant Tet M, as well as additional resistance genes. They have been found naturally in, or been introduced into, over 50 different species and 24 genera of bacteria. Recent investigations have led to insights into the molecular basis of movement of these interesting mobile elements.
Assuntos
Bactérias/genética , Conjugação Genética/genética , Elementos de DNA Transponíveis/genética , Plasmídeos/genéticaRESUMO
BACKGROUND/AIMS: Enterococci have been implicated in persistent root canal infections but their role in the infection process remains unclear. This study investigated the virulence, phenotype and genotype of 33 endodontic enterococcal isolates. METHODS: Phenotypic tests were conducted for antibiotic resistance, clumping response to pheromone, and production of gelatinase, hemolysin and bacteriocin. Genotype analysis involved polymerase chain reaction amplification of virulence determinants encoding aggregation substances asa and asa373, cytolysin activator cylA, gelatinase gelE, gelatinase-negative phenotype ef1841/fsrC, adherence factors esp and ace, and endocarditis antigen efaA. Physical DNA characterization involved pulsed-field gel electrophoresis of genomic DNA, and plasmid analysis. RESULTS: Potential virulence traits expressed included production of gelatinase by Enterococcus faecalis (n=23), and response to pheromones in E. faecalis culture filtrate (n=16). Fourteen strains produced bacteriocin. Five strains were resistant to tetracycline and one to gentamicin, whereas all were susceptible to ampicillin, benzylpenicillin, chloramphenicol, erythromycin, fusidic acid, kanamycin, rifampin, streptomycin and vancomycin. Polymerase chain reaction products encoding efaA, ace, and asa were detected in all isolates; esp was detected in 20 isolates, cylA in six isolates, but asa373 was never detected. The gelatinase gene (gelE) was detected in all isolates of E. faecalis (n=31) but not in Enterococcus faecium (n=2); a 23.9 kb deletion sequence corresponding to the gelatinase-negative phenotype was detected in six of the eight E. faecalis isolates that did not produce gelatinase. Pulsed-field gel electrophoresis and plasmid analyses revealed genetic polymorphism with clonal types evident. Plasmid DNA was detected in 25 strains, with up to four plasmids per strain and a similar (5.1 kb) plasmid occurring in 16 isolates. CONCLUSIONS: Phenotypic and genotypic evidence of potential virulence factors were identified in endodontic Enterococcus spp., specifically production of gelatinase and response to pheromones.
Assuntos
Enterococcus/patogenicidade , Pulpite/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Enterococcus/genética , Enterococcus/metabolismo , Feminino , Gelatinases/biossíntese , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Feromônios/farmacologia , VirulênciaRESUMO
Tn916 [carries tet(M)] is a 16.4-kb conjugative transposon that can establish itself in multiple copies in Enterococcus faecalis. To study the interaction of coresident homologous transposons during conjugation, an E. faecalis mutant defective in homologous recombination was utilized for construction of strains harboring Tn916 delta E (a derivative in which erm is substituted for tet) on the chromosome and Tn916 on a nonconjugative plasmid. When these strains were used as donors, the two transposons were able to transfer independently; however, they were found to transfer and become coestablished in the recipient up to 50% of the time. In contrast, cotransfer of a plasmid marker located outside the transposon occurred at a frequency of no greater than 0.5%. Separate experiments showed that mobilization of the nonconjugative plasmids pAM401 and pVA749 by chromosome-borne copies of Tn916 occurred only at low frequencies (generally less than 2% cotransfer). The data imply that the initiation of transposition of Tn916 results in a trans activation that is specific for homologous transposons present in the same cell.
Assuntos
Conjugação Genética , Elementos de DNA Transponíveis , Enterococcus faecalis/genética , Cromossomos Bacterianos/fisiologia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Escherichia coli/genética , Genótipo , Plasmídeos , Recombinação Genética , Replicon , Ativação Transcricional , Transformação BacterianaRESUMO
Members of the Tn916 family of conjugative transposons are able to insert themselves into Enterococcus faecalis hemolysin/bacteriocin plasmid pAD1 (and related elements) in such a way as to generate hyperexpression of the hemolysin/bacteriocin. To examine this phenomenon in more detail, E. faecalis (pAD1::Tn916) derivatives defective or altered in hemolysin expression were isolated and characterized with respect to production of the L (lytic) or A (activator) component (also known as CylA) and the specific location of the transposon. The mutants fell into five classes. Class 1 strains were nonhemolytic, and the related insertions mapped in a location known to affect expression of the L component. The other four classes varied from an inability to express hemolysin (class 2) to different degrees of hyperhemolytic expression (classes 3 to 5); the insertions in these classes mapped in a similar place within cylA, near the 3' end of the determinant. A previous study provided evidence that CylA is also necessary for bacteriocin immunity; however, these insertions did not destroy this function. (A Tn917 insertion in the 5' half of the determinant eliminates immunity.) In mutant classes 3 to 5, the presence of tetracycline enhanced hemolysin expression. In late-exponential-phase broth cultures, hemolysin could not be detected in supernatants of classes 2 to 5, in contrast to a wild-type control strain; however, different amounts of the L component could be detected, with the lowest in class 2 and greater-than-normal amounts in classes 3 to 5. Although nucleotide sequencing showed that the Tn916 insertions in classes 2 to 5 were at identical sites, the transposon junction sequences differed in some cases. The data indicated that cylA translation into the transposon would result in different truncation sites, and these differences were probably related to phenotype differences.
Assuntos
Enterococcus faecalis/patogenicidade , Proteínas Hemolisinas/genética , Sequência de Aminoácidos , Bacteriocinas/biossíntese , Sequência de Bases , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Enterococcus faecalis/genética , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Plasmídeos , Mapeamento por RestriçãoRESUMO
The conjugative transposon Tn916 moves intercellularly via an excision/insertion mechanism that involves products of int-Tn and xis-Tn. Tn5-insertion mutations in these genes were found to be complemented in an Enterococcus faecalis host by specific coresident transposons harboring the corresponding wild-type allele. A determinant designated traA, partially overlapping and divergently transcribed from xis-Tn, is thought to encode a key positively acting regulatory protein needed for expression of conjugation functions. This locus was also shown to express a trans-acting product.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Conjugação Genética , Elementos de DNA Transponíveis , Enterococcus faecalis/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Fímbrias , Dados de Sequência Molecular , Mutagênese InsercionalRESUMO
Transposon Tn916 is a 16.4-kilobase, broad-host-range, conjugative transposon originally identified on the chromosome of Enterococcus (Streptococcus) faecalis DS16. Its termini have been sequenced along with the junction regions for two different insertions. The ends were found to contain imperfect inverted repeat sequences with identity at 20 of 26 nucleotides. Further in from the ends, imperfect directly repeated sequences were present, with 24 of 27 nucleotides matching. The transposon junction regions contained homologous segments but of a nature not consistent with a direct duplication of the target sequence. Within the right terminus was a potential outwardly reading promoter. Tn916 is believed to transpose via an excision-insertion mechanism; based on the analyses of the termini, as well as two target sequences (before insertion and after excision), a possible model is suggested.
Assuntos
Elementos de DNA Transponíveis , Enterococcus faecalis/genética , Escherichia coli/genética , Sequência de Bases , Clonagem Molecular , Conjugação Genética , Dados de Sequência Molecular , Plasmídeos , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
The vat(D) and erm(B) genes encoding streptogramin resistance in Enterococcus faecium transferred together, and a direct physical link between erm(B) and vat(D) was detected. Both the vat(D) and erm(B) probes hybridized to fragments of different sizes in the donor and transconjugants, which indicated a transposition event.
Assuntos
DNA Bacteriano/genética , Enterococcus faecium/genética , Genes Bacterianos/genética , Metiltransferases/genética , Antibacterianos/farmacologia , Southern Blotting , Conjugação Genética , DNA Bacteriano/química , Resistência a Medicamentos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estreptograminas/farmacologiaRESUMO
Conjugative transposon Tn916 from Enterococcus faecalis DS16 encodes tetracycline resistance (Tet M) as well as determinants necessary for its own movement. Determination of the nucleotide sequence of Tn916 has been completed. The element is 18,032 bp in length and has an overall G+C content of 38.8%. Twenty-four potential open reading frames (ORFs) were identified based on sequence analysis. Similarities of the ORFs to other known determinants, which were revealed by database searches, are discussed.